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DNA conformational polymorphism  

Habilitation thesis by Lukas Trantirek, 2015

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Acknowledgements

I would like to thank all of my past supervisors and mentors who contributed to my

scientific education. In particular, I thank Prof. Jaroslav Koca and Prof. Vladimir

Sklenar, who supervised me during my undergraduate and graduate studies. I wish to

thank my postdoc supervisors, Prof. Juli Feigon and Prof. Norbert Muller, for their

support and all of the stimulating discussions we had. Additionally, I would like to

thank all of the colleagues and collaborators I had the pleasure to work with,

particularly Prof. Vacha, Prof. Sponer, Prof. Vorlickova, Prof. Fajkus, Dr.

Sychrovsky, Dr. Hansel, Prof. Dotsch, and Prof. Plavec. Special thanks belong to my

wife for all her help, support and inspiration in both my personal as well as scientific

life.

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Abbreviations

CD Circular Dichroism

CP Conformational Polymorphism

DNA Deoxyribonucleic Acid

EPR Electron Paramagnetic Resonance

FRET Förster Resonance Energy Transfer

G4 G-quadruplex

GBA Glycosidic Bond Angle

NA Nucleic Acid

NMR Nuclear Magnetic Resonance

PEG PolyEthylene Glycol

PELDOR Paramagnetic Electronic Double Resonance

RDC Residual Dipolar Coupling

XRD X-Ray Diffraction

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Table of Contents

Preface

1. Introduction 5

2. DNA conformational polymorphism 6

2.1 Conformational polymorphism due to sequence context 9

2.2 Environmentally promoted conformational polymorphism 14

2.2.1 In-cell NMR spectroscopy 18

2.2.2 In-cell EPR spectroscopy 21

2.2.3 Is the DNA inside the cells automatically in the native conformation? 22

2.2.4 In-cell single-particle FRET 23

2.2.5 Molecular basis for the modulation of the DNA structure by

environmental factors 26

2.3 Conformational polymorphism related to the kinetic control of DNA folding 30

3. Biological relevance of DNA conformational polymorphism 33

4. Future prospects 34

5. Summary 35

6. References 35

Appendix

List of publications included in the habilitation thesis 45

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Preface

This habilitation work is a compilation of selected scientific publications to which I

have contributed as the corresponding author or a co-author in the course of my

independent scientific career. These articles were published between 2005 and 2015.

A list of these publications is given on page 45. All of these publications have a

common theme related to DNA structural polymorphisms. In particular, they are

focused on the mechanistic understanding of the phenomenon of environmentally

promoted structural polymorphisms of DNA, context-dependent DNA

polymorphisms, as well as the development of novel methods to study DNA

polymorphisms. The accompanying text highlights my contribution to the field of

DNA structural biology and also contains a brief introduction to the topic.

Comprehensive information on the individual topics can be found in the enclosed

original publications. The enclosed publications also include three review articles.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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1. Introduction

Deoxyribonucleic acid (DNA) is an abundant biopolymer in all living entities, where

it functions to encode, transmit, and express genetic information. The physiological

functions of DNA are predefined by its conformational plasticity. Disturbances of the

native DNA structure correlate with dozens of pathological human conditions,

including cancer (1-5).

DNA is a fundamentally attractive drug target. The essence of the “antigene”

strategy is that it is advantageous to attack disease targets at their source, at the level

of gene expression (6,7). A protein drug target is the product of a particular gene. At

each stage of progression through the central dogma (DNA transcription to RNA and

the subsequent translation to protein), the absolute number of target molecules to be

hit by a drug inhibitor dramatically increases. A single gene makes multiple mRNA

copies, each of which is translated to make multiple copies of the target protein. The

number of target molecules is amplified at each stage in the process. By targeting the

DNA, a single gene, rather than the numerous resulting protein molecules, should

promote more selective and efficient drug action. In past two decades, various DNA

structural motifs have become valid targets for new anticancer drugs and many

leading compounds that target these motifs have entered pre-clinical or clinical trials

(8).

In addition to the biological significance of DNA, its unique nano-scale

geometry, biocompatibility, biodegradability, and molecular recognition capacity

have made it a promising candidate for the construction of novel functional nano-

materials and nano-devices (9-15). In addition, site-specific surface modification of

NAs enables the presentation of bioactive compounds at defined distances and

stochiometries, which has promoted their use in a variety of novel biomedical

applications, such as tailored cell targeting and substance delivery on demand (10,14)

DNA structure

From a chemical perspective, DNA can be regarded as a hetero-polymer composed of

four basic units (monomers), namely adenosine mono-phosphate (A), cytosine mono-

phosphate (C), guanosine mono-phosphate (G), and thymidine mono-phosphate (T).

The sequence of monomeric unit defines the DNA primary structure. In terms of its

biological function, DNA can be considered a code. While some of the DNA

functions are coded by the DNA primary structure alone, such as coding for RNA

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transcription, other DNA functions, such as those related to the regulation of gene

expression or maintenance of genome integrity, are encoded in its secondary, tertiary,

and/or quaternary structure, collectively referred to as higher-order DNA structure.

While the primary DNA code is rather simple and comprises only four “letters”, the

code of the higher order DNA structure is enormously complex. The complexity of

this code is due to the inherent structural plasticity of the DNA. In addition to the

hetero-dimeric form composed of two DNA strands (in an antiparallel orientation)

that are held together by hydrogen bonds, known as B-DNA and the predominant

structural arrangement of the genomic DNA, DNA can adopt a broad spectrum of

structural motifs. Figure 1 displays some of the basic classes of DNA motifs.

Figure 1. Schematic representations of basic DNA structural motifs: A) parallel and

antiparallel duplex, B) hairpin, C) bulge, D) junction, E) pseudo-knot, F) H-DNA, G)

kissing-hairpins, H) i-motif, and I) G-quadruplex

Importantly, a number of structurally distinct patterns exist within each of the major

classes of DNA motifs. For example, a single DNA sequence forming antiparallel

double helix can adopt at least six distinct conformations, referred to as A-, A/B-,

hybrid-, composite, B-, and Z-DNA (16).

The complexity of the DNA conformational space can be well illustrated by

the G-quadruplex motif. The G-quadruplex (also known as G4-DNA) is a four-

stranded structure formed from sequences that are rich in guanine. In G-quadruplex,

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four guanine bases align in a pseudo-plane through hydrogen-bond alignments

involving the Watson–Crick edge of a guanine and the Hoogsteen edge of its partner,

resulting in a (G:G:G:G) tetrad, as shown in Figure 2A. The quadruplex stem is

composed of stacked tetrads, with phosphodiester backbones delimiting the cavities,

termed grooves. The tetrads are held together by cations and interactions of the π

orbitals of stacked aromatic bases. Even very simple G-quadruplex-forming

sequences, such as d(G2N3)4, where N stands for an arbitrary nucleotide, can, in

principle, adopt hundereds of distinct conformations that differ in their strand

orientations, loop topologies, and/or disposition of the glycosidic bond angle of the

intervening bases, which can assume either an anti or a syn orientation (Figure 2B &

2C).

Figure 2. A) Schematic representation of the G-tetrad. B) The frame of reference for

describing quadruplex topologies. The origin, the 5’-end, sits on a strand of the

quadruplex stem progressing towards the viewer with polarity indicated by grey

ellipse on the sugar-pucker. Looping can be anticlockwise, as denoted by the arrow,

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or clockwise; (-) and (+), respectively. The description of the grooves follows

anticlockwise from first to fourth. Black and white rectangles mark G residues with

syn- and anti- glycosidic bond angle (GBA), respectively. C) All possible

combinations of GBA for (G:G:G:G) tetrads. In Ia, the definitions of medium (m),

wide (w), and narrow grooves (n) according to the adjacent disposition of GBA are

shown. D) Representations of all looping topologies possible for three loop uni-

molecular quadruplex topologies. The topologies denoted by “a” start with

anticlockwise progressing loops, and conversely the topologies denoted by “b” start

with clockwise progressing loops. The sub-figures B-D were adopted from reference

(17).

2. DNA conformational polymorphism

Conformational polymorphism is an inherent property of DNA. DNA conformational

polymorphism (CP) refers to the ability of a specific DNA sequence to exist in more

than one conformation. In structural biochemistry and biophysics, we distinguish

three main classes of conformational polymorphisms: i) sequence context-promoted

CP, ii) environmentally promoted CP, and iii) CP arising from kinetic partitioning in

the course of DNA folding. The borderlines between the individual classes are not

strictly defined and a number of DNA sequences display behavior that falls into all

three classes. It needs to be mentioned that the classes are primarily defined by the

limitations imposed by the experimental procedures used to explore the DNA

structure. The individual classes of CP and their implications for fields of structural

biology, molecular engineering and DNA drug design are discussed below.

2.1 Conformational polymorphisms due to sequence context

In eukaryotic cells, the DNA is localized in the cell nucleus and mitochondria. At

present, there is no available experimental technique that provides high-resolution

information on the DNA structure and dynamics in the context of unperturbed

genomic/mitochondrial DNA. Essentially all information on structure of biologically

interesting regions from the genomic/mitochondrial DNA comes either from X-ray

diffraction studies or solution NMR investigations conducted on model

oligonucleotides (18). The use of short model oligonucleotides presumes the

“excision” of the sequence to be studied out of the genomic context. The concept of

excision implicitly assumes that structure and dynamics of the model oligonucleotide

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are identical to that of corresponding sequences in the genome; in other words, the

structure and dynamics of the model oligonucleotide are independent of the flanking

sequence/structural context. However, a number of examples exist, where two

oligonucleotides with marginal difference in sequence (differing typically by virtue of

one or two extra flanking nucleotides) show notably different structural properties. In

this respect, we define sequence context-promoted CP as the situation when two or

more distinct “excisions” of an identical structural segment from the

genomic/mitochondrial DNA display distinct structural properties.

One of the most studied cases of sequence context-promoted CP is provided

by a DNA sequence corresponding to the 3’-G-rich single-stranded overhang (G-

overhang) from human telomeric DNA. The 50- to 200-nucleotide-long G-overhang

consists of repeating d(GGGTTA) elements (19). The G-overhang was observed to

adopt G-quadruplex (G4) structures in vitro and in vivo (20-22). A number of studies

suggested that the G-telomeric overhang consists of multiple G4 units (each unit

comprises four telomeric repeats) that are arranged in a “bead-on-the-string” fashion

(reviewed in, e.g., (22)). Folding of the G-overhang into a four-stranded G-

quadruplex has been demonstrated to inhibit telomerase, an enzyme that is activated

in more than 80% of cancers (reviewed in, e.g., (23)). As several small molecular

weight ligands that stabilize telomeric G-quadruplex structures have displayed

promising anticancer activity in tumor xenograft models, it has been proposed that

stabilization of telomeric G-quadruplexes might be applicable to the treatment of a

wide range of human cancers (reviewed in, e.g., (24,25)). However, in vivo, all of

these small molecules failed to selectively target telomeric G4-DNA relative to other

G4-forming regions in the genome (26,27). Significant interest in improving the

selectivity of small molecular weight ligands towards telomeric G4 prompted

structural studies to characterize the physiologically relevant conformation of the G4

unit within the G-overhang.

However, numerous studies using short telomeric constructs demonstrated that

the G4 conformation strongly depends on the 5’- and 3’-flanking residues

immediately adjacent to the core G4 forming sequence, namely d(GGG(TTAGGG)3)

(28). Thus far, the short oligonucleotides based on four telomeric repeat segments

were found to be capable of adopting at least four folding topologies in presence of

potassium, namely hybrid 1, hybrid 2, parallel and an anti-parallel two tetrad G4

(Figure 3), depending on the nucleotides flanking the basic G4 core (22,28-31). These

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observations raised concerns about whether the structural behavior of the G4 units

within the telomeric G-overhang might be realistically assessed in studies of short,

model telomeric sequences forming single G4 units.

Figure 3. Schematic representation of G4-DNA folding topologies observed for short

human telomeric DNA model constructs in presence of potassium, namely parallel

(32), hybrid-1 (33), hybrid-2 (34), and an anti-parallel two tetrad basket (29).

To gain insight into the conformational properties of individual G4 units

within the context of the telomeric G-overhang, we investigated site-specifically

labeled extended constructs spanning 8 and 12 telomeric repeats using high-resolution

NMR spectroscopy (35). While site-specific labeling of the construct based on 8

telomeric repeats, hereafter referred to as the double core construct, was tailored to

identify the conformation of the 3’-terminal G4 unit in the G-overhang, the specific

labeling of the construct based on 12 telomeric repeats, hereafter referred to as the

triple core construct, was adopted to provide structural information about the

conformation of the internal G4 unit(s) in the G-overhang. With this experimental

setup, we were able to show that the addition of 3’-nucleotides and the whole 5’-G4

unit to the double core construct had no significant influence on the folding topology

of the internal G4 unit. In other words, our data suggest that the 5’- and 3’-flanking-

dependent conformational polymorphisms observed for single-core G4 sequences are

diminished in long telomeric sequences that form multiple units. In addition, detailed

NMR investigations revealed that the G4 units in the G-overhang sequences coexist in

2-tetrad antiparallel basket and hybrid-2 conformations. Our investigations identified

the 2-tetrad antiparallel basket and hybrid-2 topologies as structural targets for the

development of telomere-specific ligands, while marking parallel and hybrid-1

conformations as “artifacts of the experimental design”(35). The example given above

illustrates a situation when marginal sequence variations give rise to dramatic

structural rearrangements and bias the assessment of the biological relevance of

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individual conformations within the studied DNA. In this particular case, the observed

flanking nucleotide-dependent CP for telomeric sequences forming a single G4 unit

can essentially be regarded as an artificial problem of the “inappropriate” design of

the model oligonucleotide.

Although, as demonstrated above, the sensitivity of the DNA structure to

sequence variation might impose a problem in assessing the biological relevance of

the structural data, we recently showed that it could be positively exploited to

interrogate the relationships between the DNA structure and its function, with a

number of advantages. While the precise knowledge of the G4 topology in the

telomeric G-overhang is of crucial importance for rational drug design applications,

the question is whether the physiological function of the G4 units within the G-

overhang requires a particular G4-topology. We set to address this issue by “tracing”

the phylogenetically conserved structural properties of the telomeric G-rich DNA in

the course of the evolution of eukaryotic species (36). While the function of telomeric

DNA is conserved among eukaryotes, the sequences of the telomeric DNA differ

between species. We based our analysis on the assumption that only functionally

relevant structural features are subject to evolutionary selection pressure (and are

evolutionarily conserved), while other functionally silent properties manifest

themselves as phylogenetic variables. We found that the telomeric DNA from

Caernohabditis elegans is based on a fold-back-based structure, while the telomeric

DNA from other species is based on a G4 structure. These data indicate that only the

formation of the secondary structure is required for function of telomeric DNA, but

not a particular structural type. From the point of view of biomedical applications, our

data suggest that the function of (human) telomeric DNA is not connected with any

specific G4 topology (36).

From the stereo-chemical point of view, the telomeric DNA from C. elegans

itself represents an important example of context-dependent CP. The G-rich telomeric

DNA from C. elegans, emulated by the d(GGCTTA)3GG oligonucleotide, was

previously shown to preferentially fold into a G4-quadruplex structure stabilized by

Hoogsteen base-pairs (37). However, we demonstrated that the extension of the

construct by a single 3’ nucleotide leads to the complete remodeling of the

conformation space towards the formation of the fold-back structure stabilized by

Watson-Crick base pairs (36). We could show that this fold-back structure remains in

the preferred folded topology in the extended telomeric constructs. This example also

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well illustrates problems with design of model DNA constructs: the addition/deletion

of the single nucleotide to the model construct might promote a transition between the

different structural classes.

The specific category within this class is context-dependent CP promoted by

(epigenetic) modifications of the DNA-constituting moieties, such as nucleic acid

bases or the sugar-phosphate backbone. Typical examples of such modifications are

5-methylation of cytosine residues, deamination of adenosines, or formation of abasic

sites in the polynucleotide chain (38-40). Depending on the sequence context, the

individual modifications may display a notably distinct impact on the conformational

behavior of DNA. For example, we recently showed that the generation of abasic sites

within the G4-forming sequences from human telomeric DNA are connected with the

promotion of the G4 polymorphism (41). In contrast, 5-methylation of the cytosine

residues in i-motif-forming sequences were not found to promote the polymorphisms,

but instead modulate the thermodynamic stability of the basic i-motif scaffold (42)

Together, the provided examples indicate that the basic assumption of the

excision concept in structural biology is not universally valid and that transmitting the

information on the oligonucleotide structure to its behavior in the context of the

genomic DNA needs to be done with caution. Arguably, the disregard for the context-

dependent CP is currently one of most frequent artifacts in structural biological

analysis of DNA. On a positive note, context-dependent CP can be exploited for not

only molecular engineering for the rational design of various DNA nano-machines

and devices, but also for identification of the relationships between the DNA structure

and its function.

The following articles of the applicant are related to the above topic: (K marks article corresponding author)

1: Hänsel R, Löhr F, Trantirek L, Dötsch V. High-resolution insight into G-overhang architecture. J Am Chem Soc. 2013 135(7):2816-24. IF=12.1 2K: Školáková P, Foldynová-Trantírková S, Bednářová K, Fiala R, Vorlíčková M, Trantírek L. Unique C. elegans telomeric overhang structures reveal the evolutionarily conserved properties of telomeric DNA. Nucleic Acids Res. 2015 43(9):4733-45. IF=9.1 3: Konvalinová H, Dvořáková Z, Renčiuk D, Bednářová K, Kejnovská I, Trantírek L, Vorlíčková M, Sagi J. Diverse effects of naturally occurring base lesions on the structure and stability of the human telomere DNA quadruplex. Biochimie. 2015 118:15-25. IF = 3.0

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2.2 Environmentally promoted conformational polymorphisms Due to the importance of DNA in living systems and materials science, the structures

of DNA have been the subjects of intense biophysical investigations for the last 60

years, including high-resolution methods such as x-ray diffraction (XRD) and nuclear

magnetic resonance (NMR) spectroscopy. However, instead of revealing the unique

3D structural motifs for specific DNA sequences, in many cases, these studies

provided a puzzling picture of the DNA conformational polymorphisms that appeared

to be promoted by the DNA sequence and the experimental conditions used in the

structural investigations (28,43-48).

A common property among the broad spectrum of DNA motifs that emerged

from structural investigations was the inherent sensitivity of the DNA structure and

dynamics to non-specific physical/chemical environmental factors. A number of

studies demonstrated that the stability, local DNA dynamics and the folding topology

of DNA might strongly depend on the water activity, nucleic acid concentration,

molecular crowding, pH, temperature, viscosity, or nature and concentration of the

counter ion(s) (21,28,43,45,46,48-61). The folding of the DNA G-quadruplex motifs

represents a textbook example of this type of behavior. The DNA G-quadruplexes

formed by human telomeric repeats adopt at least three distinct folding topologies and

differ in their strand polarities, loop orientations, and the orientation of the guanine

bases with respect to the sugar moiety, depending on the environmental conditions

and experimental conditions employed for their investigations (28). Hydration-

dependent A- to B-DNA helix transitions (43,55) are among the other well-known

examples of chemical/physical control over the DNA structure. Typical examples of

environmentally controlled DNA structural polymorphisms are shown in Figure 4.

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Figure 4. Example of environmentally controlled polymorphism of NA structures. A)

concentration dependent hairpin to helix transitions (61), B) pH dependent

rearrangements of i-motifs in human centromeric DNA (45,62), C) ion type,

molecular crowding, and water activity dependent polymorphism of G-quadruplex

structure (reviewed in (28)), D) Hydration-dependent A to B DNA helix transitions

(55).

From a practical point of view, we define the environmentally promoted structural

polymorphisms of DNA as DNA structural rearrangements (shifts in conformational

equilibriums) that are induced by perturbation of one or more non-specific physical-

chemical parameters in environment.

As already mentioned above and as evidenced from the structural database statistics,

essentially all of the available high-resolution information on the DNA structure

comes either from XRD or solution NMR investigations

(http://www.rcsb.org/pdb/statistics/holdings.do). It needs to be stressed that the vast

majority of these studies have been motivated by the biological relevance of the

investigated DNA motifs. However, the observation that the structural behavior of

DNA might strongly depend on the subjective choice of experimental conditions and

procedures indicates that the conventional XRD and/or NMR techniques (in fact any

in vitro spectroscopic technique) must be used with caution when addressing the

question of the physiologically relevant structure of DNA molecules. Both XRD and

NMR examine the structural properties of the isolated DNA under unnatural and

rather simplistic conditions. The environmental conditions in XRD studies are

constrained to conditions that support mono-crystal growth. In the majority of cases,

these conditions are rather artificial and involve crystallization at non-physiological

pH or in the presence of additives that facilitate the DNA crystallization process. In

addition, the crystallization process itself exposes the DNA to notable degree of

dehydration.

Considering the specifics of the XRD experimental procedure, it is of no

surprise that the DNA structures derived using XRD are often found to be distinct

from structures detected by spectroscopic methods in diluted solutions (48,52,55,61).

Nonetheless, it needs to be stressed that analyzing the DNA structure/dynamics in

solution does not necessarily ensure the physiological relevancy of the acquired

structural/dynamical information, as the choice of solution conditions is limited by the

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fact that not all of the relevant factors that can modulate the DNA structure in vivo are

known.

Influence of counter ions

A statistical analysis of the conditions used in solution NMR spectroscopy over last

30 years to determine DNA structures revealed that more than 85% of the available

structural information on DNA in solution currently comes from analyses in sodium-

based buffers (63) (Note: Sodium is a prevalent ion in the extracellular space, where

no DNA is present, while potassium is the prevalent ion in intracellular space (64)).

This analysis highlights one of the most commonly applied assumptions in solution

NMR studies that presume that the nature of the counter ion has no influence on DNA

structure and dynamics. However, shortcomings stemming from this assumption are

only now beginning to be recognized. For example, the DNA G-quadruplexes were

shown to adopt different folding topologies in the presence of sodium and potassium

(28). Ion type-dependent bending of double-stranded DNA polyA/polyT sequences

represents another known example (65). Although in a number of cases the overall

DNA structure, particularly double-stranded DNA, might be essentially identical in

both Na+ and K+-based solutions, we recently demonstrated that the local DNA

dynamics in the presence of Na+ are notably distinct from those in the presence of K+

(66).

Molecular crowding

There have been ongoing attempts to assess the physiologically relevant DNA

structure in vitro by emulating the composition of the intracellular space using

adjusted buffer compositions. In addition to salts and small molecular weight

compounds, the milieu that surrounds the chromosomes, the inter-chromatine

compartment (67), is crowded by the presence of (macro)-molecules up to ~

100mg/ml (68). A simulation of the so-called molecular crowding effect with organic

additives showed that the DNA structure is notably sensitive to crowding. The

simulated crowding effect, mostly using polyethylene glycol (PEG) 200-400, was

shown to affect the equilibrium between the DNA duplex and G-quadruplex/i-motif,

promote the inter-conversion between various G-quadruplex topologies, strengthen

the interaction between DNA and small molecular weight ligands, or promote

stabilization of DNA triplex (59,69). However, we have recently demonstrated that

the molecular crowding DNA experiences inside cells and/or in crude cellular

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homogenates is notably distinct from the conditions DNA experiences in PEG-

supplemented solutions (21,70,71). It was only recently revealed that the majority of

the observed structural phenotypes in the simulated crowded environment were due to

a direct interaction of the PEG molecules with the DNA rather than due to the

influence of molecular crowding itself (51).

Together, the experimental observations demonstrating that the folding of

NAs depends on environmental factors strongly suggest that the quantitative

characterization of physiologically relevant NA structures and dynamics should

ideally be performed either under native conditions in vivo or under in vitro

conditions that realistically emulate the complex environment of living cells. This

consideration has recently sparked interest in the development of novel tools that

allow the structural characterization of NAs in the complex cellular context.

Inception of structural analysis of NAs in living cells

The introduction of the concept of in-cell spectroscopic analysis, namely in-cell NMR

spectroscopy, in-cell EPR spectroscopy, and in-cell single particle FRET, was the first

important step towards high-resolution structural analysis of NAs in their native

environment (72-74). All of the mentioned methods are based on a similar underlying

principle, which is outlined in Figure 5. In all of the techniques, the exogenous NA

fragments are introduced into living cells, followed by the respective spectroscopic

investigations. Despite the illusion that the in vivo measurements are directly

analogous to the conventional in vitro NMR/EPR/FRET experiments, in-cell

spectroscopic analysis has developed into a separate field. The established techniques

for in vitro NMR/EPR/FRET structural analysis of NAs generally fail to provide an

expected readout under in vivo conditions due to the limited resolution and reduced

sensitivity of in-cell spectra, as well as due to the limitations imposed by biological

factors, such as cell death, accidental NA leakage from the cells, the NA cellular

localization, or degradation - for details, see (70-72). As a result, in-cell spectroscopic

analysis requires the application of unconventional approaches not only for sample

preparation and in-cell spectra acquisition but also for the interpretation of the in-cell

spectroscopic data. While in-cell spectroscopic analysis of NA structure and/or

dynamics in the native, complex cellular environment is feasible and provides

structural insights that are impossible to envision using the conventional isolated in

vitro methods, its general application remains limited due to various technical

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reasons. The limitations and application potential of the individual methods are

discussed in detail below.

Figure 5. Schematic representation of general in-cell spectroscopic setup.

2.2.1 In-cell NMR spectroscopy

Initially, in-cell NMR spectroscopy was devised to selectively observe overexpressed 13C/15N-labeled proteins in bacterial cells (75,76). Labeling with 13C/15N isotopes was

shown to be indispensable to resolve the NMR signals from the protein of interest

from the signals originating from the cellular background and to enhance the

sensitivity of the in-cell NMR detection (71,75-79). However, this approach is limited

by the requirement for high levels of the overexpressed proteins. In 2006, this concept

was extended to allow observations of proteins in eukaryotic cells, namely in Xenopus

laevis oocytes (79,80). The original and new concepts differ in the way in which the

investigated protein is deposited in the living cells. In the original approach, the

isotopically labeled protein is directly produced within the bacteria, which grows in

isotopically labeled medium. In the latter approach, the isotopically labeled protein is

not produced in oocytes, but is delivered into oocytes via microinjection. The

microinjection delivers several advantages over endogenous overexpression of the

protein. First, the deposition of the isotopically labeled protein into the isotopic label-

free cellular background allows us to monitor the introduced protein without

interference from the cellular background. Second, in contrast to the original method

based on protein overexpression, this approach can be applied to biomolecules other

than proteins.

In 2009, our group adapted the method of in-cell NMR spectroscopy to

investigate polymorphisms of nucleic acids (NAs) in the complex environment of

injected X. laevis oocytes (73). In an in-cell NMR experiment, the unlabeled or

isotopically labeled DNA fragment, which is prepared either by enzymatic or

chemical synthesis, is mechanically introduced into X. laevis oocytes via

microinjection. The intracellular concentration of the introduced DNA typically needs

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to be in the 50-250 µM range to observe and characterize the DNA structure inside a

cell. Approximately 200 oocytes need to be injected for one in-cell NMR sample. The

injected oocytes are then transferred to an NMR tube, submerged in a buffer

mimicking the composition of the extra-cellular environment and subjected to NMR

investigations (73).

In contrast to its application to proteins, the in-cell NMR of NA has identified

specific problems, such as the degradation of nucleic acids inside the living cell

(70,71,73). The DNA is exposed to nucleases when it is introduced into the cells. The

nucleolytic activity results in the relatively fast depletion of the studied NA fragment

inside the cell. The “life-span” of the DNA in vivo strongly depends on its primary

sequence and folding topology. In addition to DNA degradation, there are also other

factors that might impose additional “time” restrictions, such as leakage of introduced

DNA from cells or cell death. In general, due to the limitations arising from DNA

degradation, leakage of the DNA from cells, and/or cell death, the time-window

accessible for routine DNA exploration under in vivo conditions is typically less than

6 hours (70,71,73). This time restriction dictates the type of NMR experiments than

can be used for in vivo NA structure investigations.

Another source of restrictions in applications of in-cell NMR spectroscopy of

nucleic acids stems from the heterogeneous nature and viscosity of the intracellular

environment, as well as the heterogeneity of the NMR sample composed of stacked

oocytes. All of these factors are responsible for the generally low resolution and S/N

ratio of in-cell NMR spectra. Typically, the signals observed in the in-cell NMR

spectra are significantly broader than the signals from purified samples (70,71,73).

In general, the quantitative structure determinations using NMR spectroscopy

presume the use of isotopically labeled samples coupled with the exploitation of an

elaborate suite of hetero-nuclear NMR experiments. While there was one reported

successful protein structure determination in vivo using in-cell NMR spectroscopy

(81,82), for nucleic acids, de novo structure determination in vivo appears impractical,

although, in principle, it is feasible. The main obstacle is low in vivo stability of the

nucleic acids coupled with a prohibitively high cost for isotopically labeled samples.

An additional obstacle might be toxicity. We have observed that some NA fragments

were toxic to the injected cells at the standard concentrations required for in-cell

NMR investigations (70,71,73). Although reducing the effective concentration of the

injected NA usually diminishes toxicity, it is inherently connected with a decreased

  20  

sensitivity of the in-cell NMR experiment. As a result, the interpretation of the in-cell

NMR data adopted thus far has been primarily based on comparisons of the in vitro

and in vivo spectral fingerprints, rather than ab initio structure determination (70,71).

Comparing the spectral fingerprints of NA sequences under various in vitro

conditions that promote miscellaneous structural arrangements within the studied NA

fragments with in/ex vivo spectral patterns serves two primary purposes: i) to identify

defined in vitro conditions, mostly a specific buffer composition, to be employed for a

detailed characterization of the physiologically relevant structures using conventional

in vitro NMR spectroscopy; and ii) to identify the property of the intracellular

environment that is responsible for defining the effective conformation of the nucleic

acid in vivo. While the in vivo and ex vivo spectral patterns are recorded in living X.

laevis oocytes and/or X. laevis egg extracts, respectively, the reference in vitro data

are individually recorded at conditions simulating various degrees of molecular

crowding, viscosity, miscellaneous pH and temperature, or different compositions of a

buffer in terms of low-molecular weight compounds (21,70). In general, this approach

benefits from the high sensitivity of NMR chemical shifts to both local and global

structural changes of the nucleic acids. In principle, each NA folding topology is

marked by a unique pattern of NMR signals. As a result, an agreement between the

in/ex vivo and in vitro spectral patterns is a reliable indication of the representative in

vitro conditions under which the nucleic acid fragment adopts the same folding

arrangement as in a cell. A schematic representation of the interpretation process

based on the comparison of NMR spectral fingerprints is outlined in Figure 6.

In contrast to conventional in vitro NMR, the in-cell NMR experiment is

extremely technically challenging. The approach used to interpret the in-cell NMR

data based on comparisons between the spectral fingerprints of the NA sequences

acquired in vivo and those acquired in vitro completely fails when none of the

considered in vitro conditions provide a spectrum identical to that recorded under

native conditions in vivo. Unfortunately, these cases are biologically the most

interesting. This approach also becomes quite problematic when the in vivo and in

vitro spectra share some degree of similarity but are not entirely identical. In this case,

a philosophical problem arises in terms of the meaning of the in vitro structural data.

While the degree of spectral similarity will somehow reflect the accuracy of the

derived structural information, the precision of in the vitro-derived structure will

become meaningless in this case (please note that in XRD and in vitro NMR analyses,

  21  

the structural precision is the only measure of the structural quality). For a detailed

discussion of the application potential and limitations of in-cell NMR spectroscopy,

please refer to our recent review (70,71). Undoubtedly, the development of techniques

allowing ab initio NA structural determination in vivo is an essential prerequisite for

the general applicability of this approach and future development of the field.

Figure 6. A) and B) Outline of the interpretational process of in-cell NMR spectra

based on comparison of spectral fingerprints between in vivo and various in vitro

conditions. The in vitro conditions are employed to generate different structural

arrangements of NA fragment under the study. The figure was reproduced from our

recent review (70).

2.2.2 In-cell EPR spectroscopy

In-cell EPR spectroscopy of DNA was introduced in 2011 by Prof. Prisner and

colleagues (74). In terms of sample preparation, the in-cell EPR procedure is directly

analogous to the in-cell NMR experiment. Whereas NMR spectroscopy makes use of

the magnetic properties of the nuclear spin, EPR spectroscopy is based on the

magnetic moment of unpaired electrons. Of particular interest is a technique called

PELDOR (or DEER), which measures the dipolar coupling between unpaired

electrons that are separated by distances of 1.5–10 nm. The primary components of

nucleic acids are devoid of paramagnetic electrons; therefore, the target of interest

  22  

must be labeled with a stable paramagnetic probe. Site-directed spin labeling (SDSL)

is routinely used to attach small nitroxides, such as 3-maleimido-2,2,5,5-tetramethyl-

1-pyrrolidinyloxy (3-maleimido-PROXYL, 5-MSL) or 2,2,5,5-tetramethyl-1-oxyl-3-

methyl methanethiosulfonate (MTSL), to the NA base, sugar, or phosphate-backbone

moiety during or after chemical synthesis (50,71,74,83,84). In contrast to liquid-state

in-cell NMR spectroscopy, which is carried out at physiological temperatures, the fast

relaxation of the nitroxide spin labels at room temperature requires that the PELDOR

experiments are conducted at cryogenic temperatures (50 K), and the cells are shock-

frozen in liquid nitrogen prior to the experiment (71,85).

EPR spectroscopy cannot provide the same amount of detailed information

about the conformational and dynamic state of a macromolecule as NMR

spectroscopy. However, the advantages of in-cell EPR spectroscopy are the lack of

cellular background signals, and the possibility of measuring distances by the

PELDOR EPR method independent of the tumbling rate, and, therefore, the molecular

weight of the macromolecule (85). Instead of 200 oocytes, as required for in-cell

NMR experiments, only 30–50 injected oocytes are needed to occupy the active

volume of the EPR resonator. Thus, injection and loading can be performed within

10–15 min, while the injection of the 200 oocytes required for in-cell NMR analysis

usually takes hours.

Undoubtedly, the biggest challenge for in-cell EPR spectroscopy is the rapid

reduction of the currently available spin labels in the cellular environment, which

limits the measurement time. Igarashi et al. observed that the nitroxide spin label

attached to the target under study was quickly reduced once present in the cellular

environment, with an estimated half-life of 1 h. (86). More sterically protected spin

labels are currently being developed and will have a major impact on the application

of EPR spectroscopy in cellular systems (87) .

2.2.3 Is the DNA inside the cells automatically in the native conformation?

The “Catch-22” of all of the in vivo techniques for nucleic acid structural

characterization, including in-cell NMR and in-cell EPR, is that while these

techniques were devised for biomolecular structure analysis under native conditions,

their implementations are always connected with various degrees of disturbance of the

native environment (71,78). Without any doubt, the introduction of high

  23  

concentrations of exogenous DNA into a living cell is inherently connected with

alterations of the physico-chemical properties of the intracellular environment, which

might bias the structural readout.

While the deposition of large quantities of DNA into cells is an inherent

attribute of in-cell NMR and in-cell EPR, the cells appear to be able to diminish the

adverse effects and restore the homeostasis of their intracellular environment. There

are two options to confirm the integrity of the manipulated cells. The first is based on

a comparison of the cell viability injected with a buffer versus cells injected with a

buffer containing the DNA (70,71). The second uses the addition of progesterone to

injected oocytes and determines whether the oocytes can be driven from a G2/M

arrested oocyte to metaphase of meiosis II, i.e., to the stage of a mature egg (70,71).

As the enzymatic activities required for the transition from an oocyte to a matured egg

are sensitive to disturbances in the environment, this simple test provides a good

indication that the injected cells were able to restore the homeostasis of the

intracellular environment. A dramatic disturbance of the intracellular environment is

typically recognized within 30 minutes of the injection and is marked by increased

cell mortality.

2.2.4 In-cell single particle FRET

For both the in-cell NMR and EPR investigations, there are three principal issues that

stem from the requirement to deposit ultra-high concentrations of exogenous DNA

into the cells (73,74,84). First, as mentioned above, the introduction of high

concentrations of exogenous DNA into a living cell is inherently connected with

alterations of the physico-chemical properties of the intracellular environment, which

might bias the structural/physiologically relevant readout (78). Second, it is not only

likely that there is possible disturbance of the intracellular environment, but also the

inability to ensure that the entire introduced DNA is localized to its native, namely the

cell nucleus. Although, it was shown that small DNA fragments accumulate in the

nucleus after injection into the cytoplasm of eukaryotic cells, the localization is not

quantitative, and ~10% of the introduced DNA remains in the cell cytosol (70). Third,

our inability to deliver these high concentrations of exogenous material into bacterial

and mammalian cells limits (at the present) the in-cell NMR/EPR analysis to a single

cell type, namely X. laevis oocytes.

  24  

To resolve all of these issues, we have devised the concept of in-cell single

particle FRET of nucleic acids to allow us to characterize the DNA structure in the

complex environment of living cells at the single molecule level (72). The method is

based on the common principle of all in-cell methods: exogenous DNA is introduced

into the living cells, and, in this case, it is labeled with fluorophore tags. Quantitative

FRET analysis is then employed to convey the required structural information about

the nucleic acid fragment. The principle difference between the in-cell EPR/NMR

method and in-cell spFRET is that in in-cell spFRET, the required information on the

DNA structure is derived from measurements from a single molecule (a single

emitted photon). As the single molecule can be delivered into almost any type of

mammalian and/or bacterial cell, this method is not limited to large cells that can be

mechanically injected, such as X laevis oocytes, in contrast to in-cell NMR/EPR. The

delivery of a single molecule (or only a few molecules) into the cells has no toxic

effect (which is often observed with in-cell NMR/EPR applications), and the

disturbance of the native environment is diminished. Last, but not least, the

fluorescence from the studied molecule provides a means of verifying the DNA

localization inside the cell, and only the fluorescence from the DNA localized in the

cell nucleus can be used to quantitatively assess the DNA structure (72).

However, in terms of the data interpretation procedures, the in-cell spFRET

and in-cell EPR are essentially identical (72). The current approaches for the

interpretation of the in-cell FRET/EPR data require the existence of reference 3D

structural models. This model-based interpretation presumes that all of the possible

structural arrangements of the NAs can either be predicted or determined prior to in-

cell data interpretation using conventional approaches, such as XRD and/or NMR.

The interpretation of the EPR/FRET data is then essentially reduced to a statistical

evaluation of the closest match between the distances estimated from the set of

reference structures and the in vivo distances between either paramagnetic or

fluorescent tags acquired by in-cell EPR or in-cell FRET, respectively. This approach

has two principle weaknesses. i) It presumes the existence of accurate and precise

structural representations for the studied NA. When this requirement, which cannot be

guaranteed a priori, is not fulfilled, the procedure, which is based on an evaluation of

the closest match, will provide a “false positive” identification of the “physiological”

structure. ii) Most importantly, due to the notable uncertainty in the

paramagnetic/fluorescent tag location with respect to the NA and the altered spectral

  25  

properties of the tag in the intracellular environment (72), this approach might fail to

unambiguously discriminate even quite distinct structural motifs.

For a detail comparison of the application potential and limitations of the

individual in-cell spectroscopic techniques see (71,72).

Concluding remarks on in-cell spectroscopic methods: The development of in-cell

spectroscopic methods allowed us to characterize the DNA structure within its native

environment. The reported applications of these methods provided important insights

into the role of the intracellular environment in modulating the DNA

structure/function, and it is also important information for formulating the

composition of the buffers that are being used to emulate the parameters of the

intracellular space in in vitro spectroscopic studies. The importance of in-cell

spectroscopic methods can also be illustrated in a recent in-cell NMR study by

Selgado et al. (88), which showed that the DNA-drug interactions (drug binding

mode) observed in vivo might be distinct from those observed under artificial in vitro

conditions. This observation has profound implications for the field of drug

development, particularly for rational drug design, which entirely relies on the

knowledge of DNA structure and the DNA-drug binding modes.

However, at the same time, it needs to be stressed that the in-cell

NMR/EPR/spFRET analysis is far from being routine. The applications of these

methods are primarily hampered by technical difficulties in preparing the in-cell

samples, the high costs of the labeled material, and the limitations stemming from

problems with the acquisition and interpretation of the in-cell spectroscopic data.

Currently, only a few laboratories around the globe (less than ten) are capable of

producing and interpreting the in-cell data. It is clear than more methods should be

developed to make these techniques available to the research community. The

construction of advanced bioreactors to keep the cells alive under the harsh

experimental conditions, the development of ultra-fast schemes for the acquisition of

the in-cell NMR data, the development of interpretational schemes that allow the

determination of the ab initio structure from the in-cell NMR/EPR/spFRET data,

development of stable spin tags, development of novel approaches for geometry-

defined attachment of spin/fluorophore tags to DNA, and the development of efficient

and novel methods for the delivery of exogenous DNA into cells, among others, will

be essential.

  26  

2.2.5 Molecular basis for the modulation of the DNA structure by environmental factors The majority of studies dealing with environmentally promoted conformational

polymorphisms are primarily descriptive and limited to the identification of the

phenomena for particular DNA sequence. Only a small fraction of these studies

provides insight into the molecular mechanism by which the environmental factors

transmit their effects into the DNA structure and dynamics. Conceptually, the impact

of the water exclusion effect due to molecular crowding/confinement is well

understood: the DNA prefers to adopt conformation(s) generally marked by the

smallest hydrodynamic radius (the smaller/smallest active area for interactions with

water molecules) in environments with reduced water activity (50,53,54,56-58,89-95).

In addition to the water exclusion effect, the entropic effect is cumulative, and the

DNA disfavors extended conformations in a spatially confined environment to

minimize the collisions of the DNA with the internal surface of elastic “nano-cavity”

(60,95,96).

The DNA-ion interactions are the most problematic to study and as result the

least understood. The major impediment to the characterization of the DNA-ion

interactions, which are transient and weak in nature, is a general lack of unbiased

techniques that allow their investigation. High-resolution information on DNA-ion

interactions usually comes from either X-ray crystallography or solution NMR or

EPR analysis (97-101). In XRD studies, a low degree of hydration in the crystalline

state has been shown to change the nature of the DNA-ion interactions, favoring

direct interactions between DNA and ions over the water-mediated DNA-ion

interactions observed in solution (55). In contrast to XRD studies, NMR and EPR

measurements can be performed in a physiologically relevant hydration state.

Unfortunately, the NMR and EPR properties of the physiologically relevant counter

ions (e.g., Na+, K+, Mg2+) do not allow the application of standard techniques to

identify their interaction sites. These limitations are usually overcome by the

application of mimicking systems, referred to as “substitutionary ions” (e.g., NH4+ for

monovalent ions or Mn2+ for Mg2+) (99-101). However, the use of the substitutionary

ions brings about possible source of bias due to the differences between properties of

the native and mimicking ions. To avoid the use of the substitutionary ions, we

developed a simple approach based on monitoring the NMR cross-correlated

relaxation rates (Γ) between the aromatic carbon chemical shift anisotropy (CSA) and

  27  

the proton–carbon dipolar interaction to identify the binding sites between the

physiologically relevant counter ions and the DNA (66). The technique proved to be

extremely sensitive and can be used to detect very weak and transient interactions on

a time scale much shorter than the DNA correlation time (τc). Such interactions are

virtually invisible to conventional NMR techniques, such as chemical shift mapping

and NOESY-based techniques, which are not likely to detect ions with occupancy less

than 10% (102). Our study revealed that Na+ and K+ have distinct preferential

binding sites on DNA and that the ion binding to DNA distinctly affects the local

intra-molecular dynamics in the vicinity of the ion-coordination site(s) (66).

However, it is not just the transient and weak character of the water/ion-DNA

interactions that makes them difficult to study. As demonstrated by our studies, the

structural manifestations of the DNA water/ion interactions are often well below the

resolution of conventional NMR methods and/or schemes used to interpret the NMR

data (66,103-107). For many years, the hetero-aromatic bases, a constituent unit of

DNA, were considered to be planar and conformationally rigid (108). In 2009, our

group demonstrated that in reality, the nucleic acid bases are non-planar systems

(104). In our subsequent study, we showed that the degree of the non-planarity is fine-

tuned by the interaction of the DNA bases with the environment, represented by

networks of water molecules and ions interacting with the individual bases (106). At

the same time, our data indicated that the non-planarity is strongly correlated with the

conformation of the glycosidic bond (104,106). These data suggested that the

environment modulated the flexibility of the hetero-aromatic bases and may transmit

information about the DNA environment into the arrangement of the DNA helix

(106).

Our database study based on an inspection of the ultra-high resolution XRD

structures of DNA that revealed the details of the localized water molecules and

interacting ions indicated that the structural manifestation of the interactions between

the DNA and its environment, particularly the non-planarity of nucleic acid bases, are,

in fact, reflected in the number of observable NMR parameters, such as direct and

indirect spin-spin interactions or the NMR relaxation parameters related to local DNA

dynamics (103,105,107). However, a re-examination of the number of primary

experimental NMR data points on DNA accompanied by a series of QM calculations

revealed that the information on the environmentally dependent structural variations

is “canceled” and “spoiled” by the approximations employed in the course of the

  28  

NMR data interpretation. For example, information about glycosidic nitrogen

pyramidalization, a quantitative measure of the non-planarity of a nucleic acid base, is

canceled by inappropriate parameterization of the Karplus equation used to interpret

3JH1’-C6/8 or 3JH1’-C2/4; the corresponding parameters implicitly presume an

idealized sp2 hybridization at the glycosidic nitrogen (107). This presumption results

in over/underestimation of the glycosidic torsion angle. A similar situation holds true

for other primary NMR parameters, such as the cross-correlation relaxation rates

between glycosidic nitrogen CSA and C1’-H1’ dipole-dipole or NMR relaxation data

that depend on C6/8 and/or C1’ CSA (103,105).

The assumption of planarity of the nucleic acid bases that is implicitly

embedded in NMR data interpretational schemes not only “destroys” the information

about the interactions of individual DNA bases with the local environment but also

biases the entire structure determination process. The situation is further complicated

by errors in the empirical parameterization of the current generation of the force fields

employed in the (restrained)-MD simulations used to determine the DNA structure

from the NMR data. While the current generations of the force fields correctly

emulated the degree of nonplanarity of the nucleic acid bases, they fail to correctly

assess the chirality at the individual atoms of DNA bases (104).

Thus far, our understanding of the DNA-ion/water interactions remains

limited. The major obstacle to the characterization of the DNA-ion interactions is a

general lack of unbiased experimental techniques that allow their investigation at a

sufficient resolution.

The following articles of the applicant are related to the above topics: (K marks article corresponding author)

1K: Sychrovský V, Müller N, Schneider B, Smrecki V, Spirko V, Sponer J, Trantírek L. Sugar pucker modulates the cross-correlated relaxation rates across the glycosidic bond in DNA. J Am Chem Soc. 2005;127(42):14663-7. IF=12.1 2K: Brumovská E, Sychrovský V, Vokácová Z, Sponer J, Schneider B, Trantírek L. Effect of local sugar and base geometry on 13C and 15N magnetic shielding anisotropy in DNA nucleosides. J Biomol NMR. 2008 42(3):209-23. IF=3.1 3K: Sychrovsky V, Foldynova-Trantirkova S, Spackova N, Robeyns K, Van Meervelt L, Blankenfeldt W, Vokacova Z, Sponer J, Trantirek L. Revisiting the planarity of nucleic acid bases: Pyramidilization at glycosidic nitrogen in purine bases is modulated by orientation of glycosidic torsion. Nucleic Acids Res. 2009 37(21):7321-31. IF=9.1

  29  

4K: Hänsel R, Foldynová-Trantírková S, Löhr F, Buck J, Bongartz E, Bamberg E, Schwalbe H, Dötsch V, Trantírek L. Evaluation of parameters critical for observing nucleic acids inside living Xenopus laevis oocytes by in-cell NMR spectroscopy. J Am Chem Soc. 2009 131(43):15761-8. IF=12.1 5K: Vokácová Z, Trantírek L, Sychrovský V. Evaluating the effects of the nonplanarity of nucleic acid bases on NMR, IR, and vibrational circular dichroism spectra: a density functional theory computational study. J Phys Chem A. 2010 114(37):10202-8. IF=2.7 6K: Fiala R, Spacková N, Foldynová-Trantírková S, Sponer J, Sklenár V, Trantírek L. NMR cross-correlated relaxation rates reveal ion coordination sites in DNA. J Am Chem Soc. 2011 133(35):13790-3. IF=12.1 7K: Hänsel R, Löhr F, Foldynová-Trantírková S, Bamberg E, Trantírek L, Dötsch V. The parallel G-quadruplex structure of vertebrate telomeric repeat sequences is not the preferred folding topology under physiological conditions. Nucleic Acids Res. 2011 39(13):5768-75. IF=9.1 8: Sychrovský V, Sochorová Vokáčová Z, Trantírek L. Guanine bases in DNA G-quadruplex adopt nonplanar geometries owing to solvation and base pairing. J Phys Chem A. 2012 116(16):4144-51. IF=2.7 9K: Fessl T, Adamec F, Polívka T, Foldynová-Trantírková S, Vácha F, Trantírek L. Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET. Nucleic Acids Res. 2012 40(16):e121. IF= 9.1 10: Hänsel R, Löhr F, Trantirek L, Dötsch V. High-resolution insight into G-overhang architecture. J Am Chem Soc. 2013 135(7):2816-24. IF=12.1 11K: Hänsel R, Foldynová-Trantírková S, Dötsch V, Trantírek L. Investigation of quadruplex structure under physiological conditions using in-cell NMR. Top Curr Chem. 2013 330:47-65. IF= 5.6 6: Hänsel R, Luh LM, Corbeski I, Trantirek L, Dötsch V. In-cell NMR and EPR spectroscopy of biomacromolecules. Angewandte Angew Chem Int Ed Engl. 2014 53(39):10300-14. IF = 11.1

  30  

2.3 Conformational polymorphisms related to the kinetic control of DNA folding

CP arising due to kinetic partitioning in the course of DNA folding account for a

situation(s) when a single DNA sequence adopts two or more folding topologies,

whose populations do change over time. While this definition is general enough to

account for a simple transition from an unfolded to a folded state, the term is being

employed to denote a DNA folding process that operates on very long time scales

(hours, days, months) and along a very complex conformational surface that is

rugged, with deep competing basins of attractions.

This type of CP is characteristic for the folding of G-quadruplex-forming

sequences (44,47,109-111). In NMR, this type of CP is typically experimentally

manifested by the i) time-dependence of the NMR spectra and ii) high sensitivity of

spectral pattern to experimental (environmental) conditions including sample storage

(112). Regarding the time-dependence of the NMR spectra, the spectrum recorded

one day is notably different from the spectrum recorded on the same sample several

hours (days) later. Even for samples that were “stabilized” by so called annealing, the

procedure serves to shift the conformational equilibriums towards the most

thermodynamically stable state, and the NMR spectra are often found to change over

time. Regarding the sensitivity of the spectra to the environment, perturbations in

buffer composition have a pronounced effect on the appearance of the NMR spectra

in terms of the number of species present and their relative populations (44-

46,49,109-111,113).

This kind of behavior seems to suggest that DNA G4 folding is fundamentally

different from the funnel-like folding of small RNAs or proteins, which fold on the

time-scale of µs to ms (114,115). The folding of small proteins (or RNAs) is directed

by the formation of native, local secondary structure contacts (116). This contrasts

with the G4 DNA, where the most critical part of the folding may involve the

formation of the native, consecutive, ion-stabilized quartets. The quartets are based on

non-local interactions and, in a given fold, must have a specific combination of syn

and anti nucleotides (17,117). A decisive role of the local contacts (which would

include formation of the loop topologies) for the complete G4 fold is also not

consistent with the fact that many G4 structures fold to diverse topologies upon

changes in the environment or flanking sequences (21,44,46,49,110,113,118). In fact,

this indicates the presence of several (or even many, depending on the time

  31  

resolution) competing basins of attractions. It is likely that only a small fraction of the

molecules directly attain the most thermodynamically stable ensemble. The remaining

molecules would be first trapped in competing sub-states, i.e., misfolded states,

resulting in a multiple pathway folding process. The native and most significant

competing basins of attraction may be interchanged upon changing the experimental

conditions, which may explain why the dominant folding topology in the final

thermodynamic equilibrium is so sensitive to subtle changes in the folding conditions.

Most molecules likely initially attempt different (competing) folds and may need to

unfold to attempt another fold (112). This is entirely consistent with our simulation

studies identifying numerous highly stable potential intermediates (119,120). This has

already been visualized experimentally for the folding of the human telomeric hybrid-

1 type GQ, for which the hybrid-2 structure has been shown as a major competing

sub-state (basin of attraction) with respect to the global minimum (112). Of note, the

study by Bessi et al. (112) showed that the transition between the two folds appears to

be realized via the unfolded ensemble. It cannot be ruled out that the folding has also

been affected by other sub-states, which are already below the resolution of the

experiment. A very long time is definitely required for the most thermodynamically

stable structure (under given conditions) to be observed in the experiment.

Figure 6. Proposed mechanism for interconversion between hybrid-1 and hybrid-2

G4-DNA model constructs based on human telomeric DNA (112).

In protein or RNA structural biology, it is implicitly presumed that the biological

function is related to the lowest energy conformation. In this case, the folding times

are comparable or even below the time scale of the related biological process.

However, this assumption might not be valid for the G4 structures. Considering the

extremely slow time scale of G4 folding, which requires hours (days) to reach the

  32  

most thermodynamically stable state, it is much more likely that kinetically favored

states, which can be considered as folding intermediates, are responsible for the

biological function. This consideration implies that the G4 folding intermediates

might represent more efficient drug targets compared to the G4 thermodynamically

controlled states (121).

To gain insight into the folding pathway of the telomeric G4 structures, we

have used an MD simulation to assess the stabilities of the plausible folding

intermediates, namely G-hairpins and G-triplexes (119,120). The investigation of G-

hairpins is relevant not only to the earliest stages of the folding pathways (the hairpins

were suggested to fold on the ms time scale), but also to various later phases of

folding, as the G-hairpins may be important parts of the ensembles during the inter-

conversions among different triplex and quadruplex arrangements. Our identified

structural arrangements of G-hairpins and G-triplexes are stable enough to contribute

to the G4 folding pathways (119,120).

To date, GQ folding remains elusive, despite the intense experimental efforts

to understand the process. The basic limitation of the experimental studies is that most

of them do not allow a confident determination of the structures that are populated in

time; they are limited in terms of time, structural resolution and/or sensitivity to the

low populated species. The most recent data suggest that it may be feasible to target

the G4 folding intermediates, such as G-hairpins, G-triplexes, or kinetically controlled

G-quadruplexes, for anticancer drug designs that target G-rich regions.

The following articles are related to the above topic:

1: Stadlbauer P, Trantírek L, Cheatham TE 3rd, Koča J, Sponer J. Triplex intermediates in folding of human telomeric quadruplexes probed by microsecond-scale molecular dynamics simulations. Biochimie 2014 105:22-35. IF=3.1 2: Stadlbauer P, Kuhrova P, Banas P, Koca J, Bussi G., Trantírek L, Otyepka M, Sponer J. Hairpins participating in folding of human telomeric sequence quadruplexes studied by standard and T-REMD simulations. Nucleic Acids Res. 2015, in press(gkv994). IF=9.1

  33  

3. Biological relevance of environmentally controlled DNA polymorphisms

The sensitivity of the DNA structure and dynamics to non-specific physical/chemical

factors is an inherent property of DNA. While this phenomenon is being heavily

exploited in molecular engineering to construct dynamic DNA assemblies, such as

logical gates, conformational switches, nano-machines, and/or biosensors (122-124),

it remains unclear whether this phenomenon is, in fact, biologically relevant.

Although a number of DNA related processes, such as those connected to changes in

gene/ncRNA expression, are accompanied by natively occurring fluctuations in the

composition and properties of the intracellular space, such as pH, [Ca2+], [K+]/[Na+]

fluctuations in the course of cell- cycle, stress responses like hypoxia or hyperthermia,

apoptosis, or the degree of molecular crowding/confinement in the course of

chromatin remodeling (68,125-129), the direct link between environmentally

controlled DNA polymorphisms and these environmental changes/biological process

has not yet been demonstrated. One of the plausible explanations might be that within

the ranges of the environmental conditions tolerated by the mechanisms controlling

cellular homeostasis, the phenomenon of environmentally controlled DNA

polymorphisms does not manifest itself and/or is simply functionally silent in vivo.

Similar considerations do apply to the DNA structural polymorphisms that

arise from kinetic control of DNA folding. As shown for the G-quadruplexes formed

from human telomeric DNA repeats, certain G4 topologies are kinetically favored

over the topology corresponding to the lowest energy conformation (111,112).

However, in this case, the manifested polymorphism is “temporary”. In the initial

stages of the folding process, the polymorphic mixture consists of a small fraction of

the slowly formed, most thermodynamically stable species and a predominant fraction

of the (multiplex) kinetically favored ones. The ratio between the kinetically and

thermodynamically favored species changes over time. In thermodynamic

equilibrium, the polymorphism is essentially diminished and G4 adopts a topology

corresponding to the most thermodynamically stable conformation. The kinetically

promoted polymorphism needs to be considered because G4 folding operates on an

extremely slow time scale, which, in certain cases, may take months or even years.

However, what is the biological relevance of this kinetically induced polymorphism?

Biological processes take place at millisecond-to-second time scales. As result, the

thermodynamically favored structure is unlikely to participate in the related biological

  34  

process. The complexity of the folding pathway indicates that a number of

conformationally distinct species co-exist, even in the initial stage of the folding

process. It is still an open question whether any particular conformation from the

kinetically controlled mixture bears the function, or whether an equilibrium among

the co-existing species is important for the function.

4. Future prospects

Most of the current research interest about DNA relates to its conformational

polymorphisms. In materials science, the DNA conformational polymorphisms are

being exploited for the construction of a controllable assembly of static structures,

such as one-dimensional nanowires and 3D nano-structures (DNA origami); macro-

sized materials, such as DNA hydrogels; or the construction of dynamic assemblies,

including molecular motors, biosensors, fabricated nano-containers for controlled

release (smart drug delivery systems), molecular logic gates, unidirectional DNA-

walkers, surfaces for reversible cell adhesion; and reversible systems for the

aggregation and dispersion of carbon nano-materials or hydrophobic dendrimers. In

addition to the nano-technology applications, DNA has also recently regained

attention as an attractive drug target in a wide variety of human pathological

conditions, particularly due to the increasing functional importance of G4 structures

for the regulation of gene expression and/or maintenance of genome integrity.

Development in these fields strongly relies on our understanding of the DNA

structure/dynamics.

Despite all of these intense efforts, our understating of some of the

fundamental DNA properties, particularly DNA folding and its response to non-

specific physical-chemical factors, remains limited. Undoubtedly, the major

impediment to our understanding is a general lack of unbiased techniques that allow

their investigation at a sufficient temporal resolution and in the complex environments

of living cells. However, recent advances and the development of novel tools and

methods for studying the DNA structure, such as in-cell spectroscopic techniques that

allow us to investigate DNA structure and its interaction with drug-like molecules in

the physiological environment, holds promise for the future.

  35  

5. Summary

Deoxyribonucleic acid (DNA) is abundant biopolymer in all living entities. In terms

of biological function, DNA can be considered a code. While some of the DNA

functions are coded by the DNA primary structure alone, such as RNA transcription,

other DNA functions, such as those related to the regulation of gene expression or the

maintenance of genome integrity, are encoded in its secondary, tertiary, and/or

quaternary structure. While the primary DNA code, is rather simple and comprises

only four “letters”, the code of the higher order DNA structure is enormously

complex. The complexity of this code is due to the inherent structural plasticity of the

DNA that is finely tuned by a variety of non-specific environmental factors and/or

specific interactions, such as those between DNA and DNA-binding proteins. In this

habilitation thesis, I outline a role for non-specific physical-chemical factors in

remodeling the DNA conformational space. The habilitation thesis summarizes my

contribution to the field of DNA structural biology. In particular, the thesis

recapitulates my contribution to the mechanistic understanding of the phenomenon of

environmentally promoted and context-dependent structural polymorphisms of DNA

and also recaps my contribution to the development of methods to investigate the

DNA structure in the complex environment of living cells.

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APENDIX List of publications of the applicant included in this habilitation work

K marks article corresponding author

1: Stadlbauer P, Kuhrova P, Banas P, Koca J, Bussi G., Trantírek L, Otyepka M,

Sponer J. Hairpins participating in folding of human telomeric sequence quadruplexes

studied by standard and T-REMD simulations. Nucleic Acids Research, accepted, pii:

gkv994. IF=9.1

2K: Školáková P, Foldynová-Trantírková S, Bednářová K, Fiala R, Vorlíčková M,

Trantírek L. Unique C. elegans telomeric overhang structures reveal the

evolutionarily conserved properties of telomeric DNA. Nucleic Acids Res. 2015

43(9):4733-45. IF=9.1

3: Konvalinová H, Dvořáková Z, Renčiuk D, Bednářová K, Kejnovská I, Trantírek L,

Vorlíčková M, Sagi J. Diverse effects of naturally occurring base lesions on the

structure and stability of the human telomere DNA quadruplex. Biochimie. 2015

118:15-25. IF=3.0

4: Stadlbauer P, Trantírek L, Cheatham TE 3rd, Koča J, Sponer J. Triplex

intermediates in folding of human telomeric quadruplexes probed by microsecond-

scale molecular dynamics simulations. Biochimie 2014 105:22-35. IF=3.0

5: Hänsel R, Luh LM, Corbeski I, Trantirek L, Dötsch V. In-cell NMR and EPR

spectroscopy of biomacromolecules. Angewandte Angew Chem Int Ed Engl. 2014

53(39):10300-14. IF=11.1

6: Hänsel R, Löhr F, Trantirek L, Dötsch V. High-resolution insight into G-overhang

architecture. J Am Chem Soc. 2013 135(7):2816-24. IF=12.1

7K: Hänsel R, Foldynová-Trantírková S, Dötsch V, Trantírek L. Investigation of

quadruplex structure under physiological conditions using in-cell NMR. Top Curr

Chem. 2013 330:47-65. IF= 5.6

8K: Fessl T, Adamec F, Polívka T, Foldynová-Trantírková S, Vácha F, Trantírek L.

Towards characterization of DNA structure under physiological conditions in vivo at

the single-molecule level using single-pair FRET. Nucleic Acids Res. 2012

40(16):e121. IF= 9.1

  45  

9: Sychrovský V, Sochorová Vokáčová Z, Trantírek L. Guanine bases in DNA

G-quadruplex adopt nonplanar geometries owing to solvation and base pairing. J

Phys Chem A. 2012 116(16):4144-51. IF=2.7

10K: Fiala R, Spacková N, Foldynová-Trantírková S, Sponer J, Sklenár V, Trantírek

L. NMR cross-correlated relaxation rates reveal ion coordination sites in DNA. J

Am Chem Soc. 2011 133(35):13790-3. IF=12.1

11K: Hänsel R, Löhr F, Foldynová-Trantírková S, Bamberg E, Trantírek L, Dötsch V.

The parallel G-quadruplex structure of vertebrate telomeric repeat sequences is

not the preferred folding topology under physiological conditions. Nucleic Acids

Res. 2011 39(13):5768-75. IF=9.1

12K: Vokácová Z, Trantírek L, Sychrovský V. Evaluating the effects of the

nonplanarity of nucleic acid bases on NMR, IR, and vibrational circular dichroism

spectra: a density functional theory computational study. J Phys Chem A. 2010

114(37):10202-8. IF=2.7

13K: Sychrovsky V, Foldynova-Trantirkova S, Spackova N, Robeyns K, Van

Meervelt L, Blankenfeldt W, Vokacova Z, Sponer J, Trantirek L. Revisiting the

planarity of nucleic acid bases: Pyramidilization at glycosidic nitrogen in purine bases

is modulated by orientation of glycosidic torsion. Nucleic Acids Res. 2009

37(21):7321-31. IF=9.1

14K: Hänsel R, Foldynová-Trantírková S, Löhr F, Buck J, Bongartz E, Bamberg E,

Schwalbe H, Dötsch V, Trantírek L. Evaluation of parameters critical for observing

nucleic acids inside living Xenopus laevis oocytes by in-cell NMR spectroscopy. J

Am Chem Soc. 2009 131(43):15761-8. IF=12.1

15K: Brumovská E, Sychrovský V, Vokácová Z, Sponer J, Schneider B, Trantírek L.

Effect of local sugar and base geometry on 13C and 15N magnetic shielding

anisotropy in DNA nucleosides. J Biomol NMR. 2008 42(3):209-23. IF=3.1

16K: Sychrovský V, Müller N, Schneider B, Smrecki V, Spirko V, Sponer J,

Trantírek L. Sugar pucker modulates the cross-correlated relaxation rates across the

glycosidic bond in DNA. J Am Chem Soc. 2005;127(42):14663-7. IF=12.1